Neuromedin B is the only known mammalian peptide of the ranatensin/litorin subgroup of the bombesin family and contains 70% homology with bombesin. The three amino acid substitutions are sufficient to provide the peptide with a unique set of physiological characteristics. Neuromedin B administered icv in pylorus-ligated rats failed to reduce gastric secretion even though it is a true neuropeptide with wide distribution in brain tissue. In extracts of dissected brain regions, the peptide was found by radioimmunoassays to be widely distributed: the pituitary gland (posterior lobe greater than anterior portion), the limbic system (hypothalamus greater than hippocampus greater than thalamus), and other brain regions (caudate, cerebral cortex, substantia nigra, brain stem). These results were further confirmed by the immunohistochemical localization of neuromedin B in nerve cells throughout rat brain. Preliminary data indicate the presence of a neuromedin B staining cell layer in the coronal region of the cerebral cortex with scattered cells in the underlying layers, individuals cells dispersed among the pyramidal region of the hippocampus, a nucleus in the ventromedial hypothalamus, and dispersed cell bodies with long axons in the reticular net of the brain stem. Application of four polyclonal antibodies revealed that the amount of peptide detected depends upon the site-directed specificity of a particular antibody as well as the extraction conditions employed. Isocratic HPLC further revealed that each polyclonal antibody recognized different molecular forms of immunoreactive neuromedin B in rat spinal cord. Data also indicate that neuromedin B levels in the pituitary and limbic region decrease dramatically in lactating females and rise slowly during weaning, except in the hypothalamus which remains at the depressed state. In toto, the evidence suggests that pattern and distribution of neuromedin B defines a new neuronal pathway which may be involved in a regulatory capacity in the autonomic nervous system and subject to hormonal control.